CN112410350B

CN112410350B - Upland cotton GhWRKY74 protein and coding gene and application thereof

Info

Publication number: CN112410350B
Application number: CN202011470720.6A
Authority: CN
Inventors: 简桂良; 贺浪; 司宁
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-09-14
Anticipated expiration: 2040-12-14
Also published as: CN112410350A

Abstract

The invention discloses upland cotton GhWRKY74 protein and a coding gene and application thereof. The cDNA full-length nucleotide sequence of the gene is shown as SEQ ID No. 1; the amino acid sequence is shown in SEQ ID No. 2. The gene GhWRKY74 related to the verticillium wilt is obtained by cloning from 86-1 leaves of a verticillium wilt-sensitive cotton variety, the RT-PCR confirms that the gene is expressed synergistically in roots, stems and leaves under the stress of verticillium wilt bacteria, and the VIGS silences the expression of the gene, so that the verticillium dahliae resistance of the verticillium wilt-sensitive upland cotton variety can be improved, the verticillium wilt resistance of the upland cotton is improved, and a theoretical basis is provided for genetic improvement of the verticillium wilt-sensitive upland cotton variety.

Description

Upland cotton GhWRKY74 protein and coding gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to upland cotton GhWRKY74 protein and a coding gene and application thereof.

Background

Cotton is an important fiber crop and oil crop in the world, and cotton is planted in 24 out of 31 provinces in China, wherein nearly 3 hundred million ginseng is produced with the cotton, and 30 percent of the total sowing area is used for cotton planting. Among them, Verticillium wilt (Verticillium wilt) has serious harm to cotton yield and fiber quality in production, and the Verticillium wilt harms the vascular bundle of cotton, so that chemical agents are difficult to control, and the most effective control way is to cultivate and plant disease-resistant varieties at present. In the past, the research on the gene related to the verticillium wilt resistance of upland cotton is slow, and the major gene for verticillium wilt resistance is not reported. Because of the changeability of verticillium wilt, the disease resistance of disease-resistant varieties cultivated in the past is lost and cannot be endured, a series of disease-resistant varieties such as Acala, Sicala 1 and the like are cultivated in sequence in countries such as the United states, Australia and the like, but the varieties can only reach the disease resistance level in China, and the gene Ve for resisting verticillium wilt is cloned from tomatoes by Kawchuk, but the gene Ve can only mediate the resistance to the small variety of verticillium dahliae 1, and the resistance has specificity. At present, most of the research is devoted to the discovery of disease-resistant genes, while the research on disease-sensitive genes (S genes) is ignored. In fact, plant disease resistance and disease susceptibility are opposite, and resistance can be obtained by mutating disease susceptibility gene.

The definition of the S gene refers to a gene that promotes pathogen infestation and facilitates affinity interactions in plants. When the S gene is mutated, the plant no longer supports pathogen-affinity interaction, and thus acquires specific resistance. Plant S genes can be classified into the following types according to the different stages of the plant-pathogen interaction process: (1) genes that allow host-pathogen interaction, facilitating pathogen recognition and infection; (2) a gene encoding for down-regulation of an immune signal; (3) after infection, the genes meet the metabolism or structure requirements of pathogens and proliferate.

WRKY transcription factors (WRKY transcription factors) are plant-specific transcription factors, contain zinc finger structures and highly conserved WRKY structural domains at the C-terminal of the WRKY transcription factors, and are classified according to the number of the WRKY structural domains and the characteristics of the zinc finger structures, namely three groups I, II and III. Wherein group I is C2H2 type (C-X4-5-C-X22-23-H-X1-H), group II is the same as group I, and group III is C2HC type (C-X7-C-X23-H-X1-C). In past researches, WRKY transcription factors are found to be a huge family, and 102 WRKY genes are identified in rice and have the functions of transferring defense signals and preventing pathogenic bacteria from invading. WRKY transcription factor can be positively and negatively regulated on host defense, most of past researches are focused on positive disease resistance reaction participated by WRK gene, Wang and the like find that AtWRKY53 and AtWRKY70 positive regulation System Acquired Resistance (SAR) in Arabidopsis thaliana; deslandes et al found that AtWRKY52 includes structural features of the nucleotide-binding-Leu-rich repeat type R gene product and the WRKY domain, which is broadly resistant to bacterial wilt (Ralstonia solanacearum); yang and the like find that 11 WRKY genes such as WRKY 1, WRKY 3, WRKY5 and the like have positive regulation effect in the resistance reaction of the potatoes to late blight pathogen infection; however, since the twenty-first century, many researchers turned to the negative regulation effect of WRKY-TF on pathogenic bacteria infection, Grunewald et al found that WRKY23 transcription factor is expressed at the early stage of establishment of nematode feeding site, and the knock-out WRKY23 can reduce invasion of cyst nematode (Heterodera schachtii); the rice OsWRKY76 and OsWRKY45-1 gene overexpression plants show higher sensitivity, and the knockout plants of OsWRKY45-1 show enhanced resistance to rice bacterial blight (Xanthomonas oryzae); the CaWRKY58 gene of pepper is located in the cell nucleus, and in pepper plants infected with Ralstonia solanacearum, tobacco plants over-expressing CaWRKY58 show more serious disease symptoms than wild plants. CaWRKY58 pepper plants silenced by virus-induced gene silencing (VIGS) showed enhanced resistance to virulent ralstonia solanacearum strain FJC100301, indicating that CaWRKY58 acts as a transcriptional activator of negative regulators in resistance of pepper to ralstonia solanacearum infection. A recent study shows that a III-group WRKY transcription factor GhWRKY70 in upland cotton negatively regulates the infection of verticillium dahliae in at least two ways. And how to screen more transcription factors capable of differentially expressing WRKY so as to provide candidate genes for cultivating cotton verticillium wilt resistant varieties and have important significance for improving the existing poor verticillium wilt resistance condition of cotton.

Disclosure of Invention

The invention aims to provide upland cotton GhWRKY74 protein and a coding gene and application thereof, and aims to solve the problems in the prior art, VIGS proves that the upland cotton GhWRKY74 protein has an important role in verticillium wilt resistance, is synergistically expressed in roots, stems and leaves of high-susceptibility verticillium wilt varieties, and has the capability of reducing verticillium wilt according to verticillium wilt resistance evaluation, and silencing can improve verticillium wilt resistance of high-quality high-yield susceptible cotton varieties.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a gene GhWRKY74 related to greensickness induced by upland cotton, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.

Preferably, the primer for cloning the GhWRKY74 is shown as SEQ ID NO: 3-SEQ ID NO: shown at 13.

The invention also provides a protein coded by the upland cotton verticillium wilt related gene GhWRKY74, wherein the amino acid sequence of the protein is shown as SEQ ID NO:2, respectively.

Further, the protein also comprises a sequence shown as SEQ ID NO:2 is substituted, deleted or added with one or more amino acids, and has a protein mutation sequence for improving the verticillium wilt resistance of upland cotton.

The invention also provides application of the upland cotton verticillium wilt-resistant related gene GhWRKY74 in verticillium wilt resistance, and the gene expression is reduced by silencing the GhWRKY74 gene through VIGS, so that the verticillium wilt resistance of upland cotton varieties with verticillium wilt is improved.

The invention also provides application of the protein coded by the upland cotton verticillium wilt related gene GhWRKY74 in verticillium wilt resistance, and the gene expression is reduced by VIGS silencing GhWRKY74 gene, so that the verticillium wilt resistance of upland cotton varieties with verticillium wilt is improved.

The invention discloses the following technical effects:

the invention discloses a upland cotton verticillium wilt-resistant related gene GhWRKY74, which is a GhWRKY74 gene differentially expressed and screened according to the upland cotton transcriptome sequencing result, wherein the expression mode of the gene is down-regulated expression, and the gene is subjected to function verification through clone analysis of the gene and application of virus-induced gene silencing (VIGS) technology to confirm the function of the gene in verticillium wilt resistance in upland cotton, so that the gene can provide a candidate gene for cultivating verticillium wilt-resistant varieties of crops such as cotton.

The invention also provides a protein coded by the upland cotton verticillium wilt-resistant related gene GhWRKY74, the GhWRKY74 gene related to the upland cotton verticillium wilt resistance is obtained by cloning from cotton leaves, RNA of different tissues of roots, stems and leaves of cotton verticillium wilt-resistant varieties is extracted, RT-PCR is carried out to prove that the expression is carried out on the roots, stems and leaves of the cotton verticillium wilt-resistant varieties, and the relative expression amount in the stems is 2.8 times that in the leaves and 7.6 times that in the roots. According to the evaluation of verticillium wilt resistance, the verticillium wilt resistance is proved to have the capability of reducing verticillium wilt, and the verticillium wilt resistance of high-quality and high-yield susceptible cotton varieties can be improved by silencing. The gene GhWRKY74 provides scientific basis for applying the gene GhWRKY74 to genetic breeding of upland cotton and other crops.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a graph comparing the disease resistance difference of upland cotton treated differently in example 4; analyzing the function of the gene through virus-induced gene silencing, wherein Cotton shrink virus (CLCrV) silencing vectors pCLCrVA and pCLCrVB are presented by professor Zhouyouping snow-hei of plant protection research institute of Chinese academy of agricultural sciences, WT and WT + V991 are Cotton planted in wild type verticillium wilt-sensitive upland Cotton varieties of 86-1 before and after inoculation respectively; pCLCrV (A + B) and pCLCrV (A + B) + V991 are cotton plants 86-1 in greensickness-sensitive upland cotton varieties which are transferred into an empty carrier before and after inoculation respectively; pCLCrVA-GhWRKY74-pCLCrVB and pCLCrVA-GhWRKY74-pCLCrVB + V991 are respectively cotton plants 86-1 in greencotton varieties with greensickness of silencing GhWRKY74 genes before and after inoculation;

FIG. 2 shows the result of bioinformatic analysis of the GhWRKY74 protein; a is the amino acid composition of GhWRKY74 protein; b is a phylogenetic tree of GhWRKY74 and WRKY74 of different species;

FIG. 3 is an amino acid comparison and phylogenetic analysis of GhWRKY74 and Arabidopsis WRKY transcription factor; a is the amino acid sequence comparison of arabidopsis II d class WRKY TFs and GhWRKY 74; b is phylogenetic analysis of GhWRKY74 and Arabidopsis WRKY TFs;

FIG. 4 shows the qRT-PCR technology for detecting the expression level of target gene; pCLCrV (A + B) is a cotton plant containing an empty vector; pCLCrVA-GhWRKY74-pCLCrVB is a cotton strain containing a carrier for silencing GhWRKY74 gene;

FIG. 5 shows RT-PCR verification of relative expression levels in roots, stems and leaves of verticillium wilt-sensitive cotton varieties;

FIG. 6 is an electrophoretic map of the full length of the cDNA sequence of gene GhWRKY 74.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 cloning of upland cotton-induced greensickness-associated Gene GhWRKY74

The method for cloning the upland cotton verticillium wilt-related gene GhWRKY74 comprises the following steps:

extraction of RNA

Respectively extracting the RNA of a leaf sample of a cotton variety 86-1 by using an RNAprep Pure plant polyphenol polysaccharide total RNA extraction kit.

Synthesis of cDNA

2.1 Synthesis of intermediate fragment cDNA

Synthesis of intermediate fragment cDNA was reverse transcribed using the FastQuant cDNA first strand synthesis kit.

2.23' Synthesis of cDNA

The synthesis system of 3' -terminal cDNA is shown in Table 1 below:

TABLE 1

Mixing the above systems, centrifuging, placing on PCR instrument at 42 deg.C for 60min and 70 deg.C for 15min, cooling on ice after reaction, and storing at-20 deg.C.

2.35' Synthesis of cDNA

The synthesis of 5' RACE cDNA was as follows:

the first step is as follows: adding the reagents shown in Table 2

TABLE 2

Mix well, centrifuge briefly and place on ice.

The second step is that: adding the reagents shown in Table 3

TABLE 3

After mixing well, the 11 μ L product was placed in a PCR instrument and the reaction was programmed: 72 ℃ for 3min and 42 ℃ for 2 min. After finishing, cooling for 1min for standby.

The third step: adding the reagents shown in Table 4

TABLE 4

Mixing, and centrifuging for a short time.

The fourth step: adding the reagents shown in Table 5

TABLE 5

Gently pipette with pipette gun, mix well, and centrifuge briefly. Placing in a PCR instrument, setting a program: 90min at 42 ℃ and 10min at 70 ℃. The cDNA obtained by the reaction was diluted with an appropriate amount of Tricine-EDTA Buffer and stored at-20 ℃.

3. Primer design

RACE primers (Table 6) are designed from primer 5.0, GSP and UPM primer kits are provided, and the designed primers are sent to the synthesis.

TABLE 6 RACE primers

full-Length clone GhWRKY74

4.1 cloning of intermediate fragments of the Gene of interest

Based on the known cDNA fragments, the intermediate primers were designed and mixed in the following system (Table 7) to perform PCR amplification.

TABLE 7

PCR procedure: 3min at 94 ℃; 30s at 94 ℃,30 s at 58 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃; storing at 4 ℃. The amplification products were analyzed by agarose gel electrophoresis.

4.2 cloning of the 3' end of the Gene of interest

3' RACE is amplified by a nested PCR method,

first round PCR amplification system for 3' RACE (table 8):

TABLE 8

Mixing the above systems, centrifuging for a short time, and performing PCR amplification.

PCR procedure: 3min at 94 ℃; 30s at 94 ℃,30 s at 55 ℃, 2min at 72 ℃ and 20 cycles; 10min at 72 ℃; storing at 4 ℃. The amplification products were analyzed by agarose gel electrophoresis. Then, the first round PCR amplification product is diluted by 50 times, and the second round PCR amplification is carried out.

Second round PCR amplification system for 3' RACE (table 9):

TABLE 9

PCR procedure: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s,55 ℃ for 30s, and 72 ℃ for 1 min; 10min at 72 ℃; storing at 4 ℃. And (3) analyzing the amplified product by agar gel electrophoresis, then carrying out gel recovery, connection and transformation, and picking positive clones for sequencing.

4.3 cloning of the 5' end of the Gene of interest

After diluting the 5 'RACE cDNA obtained by the reaction with a proper amount of Tricine-EDTA Buffer, the PCR amplification of the 5' RACE is carried out, and the PCR system is as follows:

step 1 addition of reagents shown in Table 10

Watch 10

Mix gently, centrifuge briefly, and place on ice.

step 2 addition of reagents shown in Table 11

TABLE 11

After the system was prepared according to the above procedure, it was gently mixed, centrifuged briefly, and PCR amplification was performed according to the following procedure.

PCR procedure (see table 12):

TABLE 12

After the reaction is finished, performing gel agarose electrophoresis analysis, observing the band condition, and if a dispersion band or no band appears, performing the following operations:

(1) the template is a 50-fold dilution product (Tricine-EDTA buffer) of the PCR amplification product

(2) The primers are UPMS and 5' IGhWRKY74, each of which is 1. mu.L, the PCR system is adopted, and the reaction program is set as follows: 3s at 94 ℃,30 s at 65 ℃, 1min at 72 ℃ and 20 cycles; storing at 4 ℃.

After the reaction is finished, gel agarose electrophoresis analysis is carried out, then gel is recovered, connected and transformed, and positive clones are picked for sequencing.

4.4 full-Length GhWRKY74 clone

The intermediate fragment, the 3 'RACE fragment and the 5' RACE fragment are spliced through DNAman, a full-length primer qGhWRKY74-F, qGhWRKY74-R (table 6) of GhWRKY74 is designed, and full-length PCR amplification is carried out. The 5' synthesized cDNA was selected and diluted 5-fold with Tricine-EDTA Buffer as template.

The reaction system is shown in table 13:

watch 13

PCR procedure: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s,55 ℃ for 30s, and 72 ℃ for 2 min; 10min at 72 ℃; storing at 4 ℃. The amplification products were analyzed by agarose gel electrophoresis, gel recovery was performed, and A tail was added, as shown in Table 14 below:

TABLE 14

Reaction conditions are as follows: 30min at 72 ℃.

The full-length PCR reaction solution is connected to a T1 simple vector, transformed into Escherichia coli DH5 alpha, and positive clones are picked and sent to the worker for sequencing.

The results are shown in figure 6: the full length of the cDNA sequence of the gene GhWRKY74 is 1786bp, 5 'end UTR (untranslated region) 291bp, 3' end UTR 514bp and Open Reading Frame (ORF)995bp, and the sequence is shown as SEQ ID No. 1.

Example 2 bioinformatic analysis of GhWRKY74 protein

Predicted by an on-line tool ProtParam, the molecular weight of the protein is 36.82626kDa, the isoelectric point is 9.80, and the molecular formula is C₁₆₀₆H₂₅₆₄N_478O472S₂₂(ii) a Instability index 56.41, belonging to unstable proteins; the total average hydrophilicity is-0.653, and belongs to hydrophilic protein; the 20 amino acids that make up the protein, serine (Ser), account for the highest percentage (13.8%) (as shown in figure 2A), and contain 23 negatively charged amino acid residues, and 47 positively charged amino acid residues.

Using SignalP 4.0 to predict that the protein has no signal peptide; TMHMM Server v.2.0 predicts no transmembrane helical region of the protein; the CDD predicted protein has a zinc finger domain and a WRKY domain.

Through multi-sequence comparison and MEGA software, BoostStrap is set as 1000 to construct a phylogenetic tree (shown in figure 2B) of GhWRKY74, which shows that the protein similarity of GhWRKY74 with woody cotton (Gossypium arboreum) and Gossypium raimonidii (Gossypium raimonidii) is highest and the genetic relationship is closest; and cocoa (Theobroma cacao), mallow Columbia (Herrania officinalis) and durian (Durio zibethinus) form one branch of the evolutionary tree.

The phylogenetic tree was constructed using the maximum likelihood method in MEGA 5.0. By constructing a phylogenetic tree of GhWRKY74 and Arabidopsis thaliana WRKY family members (as shown in figure 3), the GhWRKY74 belongs to II d group WRKY members, contains a WRKY structural domain and a zinc finger structure of a C2H2 mode (CX (4-5) CX (22-23) HXH), and has a nuclear localization signal KRRK.

Example 3 analysis of expression levels of GhWRKY74 in different tissues of cotton susceptible variety 86-1

3.1 Experimental methods:

when upland cotton 86-1 is cultured until 2 true leaves are unfolded, respectively taking root, stem and leaf of cotton, fully cleaning and surface sterilizing, freezing with liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 deg.C.

Respectively extracting RNA of different cotton tissue samples by using an RNAprep Pure plant polyphenol polysaccharide total RNA extraction kit, carrying out reverse transcription on cDNA by using a FastQuant cDNA first strand synthesis kit, and measuring the expression quantity of the GhWRKY74 gene in different tissues after the reverse transcription. (reagent kit purchased from Beijing Tiangen)

3.2 Experimental results:

by extracting RNA of different tissues of roots, stems and leaves of cotton susceptible varieties, RT-PCR proves that the RNA is expressed in the roots, stems and leaves of cotton susceptible to verticillium wilt, and the relative expression quantity in the stems is 2.8 times of that in the leaves and 7.6 times of that in the roots (as shown in figure 5)

Example 4 Verticillium dahliae stress silenced verticillium wilt-susceptible upland cotton variety 86-1 in its verticillium disease resistant state

4.1 Experimental methods:

the target gene was cloned according to the primer VIGS-GhWRKY74-F, VIGS-GhWRKY74-R (Table 16), and the reaction system is shown in Table 15 below:

watch 15

PCR procedure: 3min at 94 ℃; 30s at 94 ℃,30 s at 55.8 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃; storing at 4 ℃.

TABLE 16 VIGS primers

Analyzing the amplified product by agar gel electrophoresis and recovering a target fragment, carrying out double enzyme digestion on the amplified product and a VIGS silencing vector pCLCrVA vector by using SpeI and PacI, connecting the recovered product and the VIGS silencing vector pCLCrVA vector by using T4 ligase and transforming Escherichia coli DH5 alpha, transforming the correctly identified GhWRKY74 silencing vector into agrobacterium EHA105 after colony PCR and double enzyme digestion verification, and adding glycerol into successfully transformed agrobacterium liquid to store at-80 ℃ after the colony PCR identification.

Cotton planting: the cotton seeds are delinted by concentrated sulfuric acid, full seeds with consistent sizes are selected for subsequent tests, the seeds are soaked in 70% ethanol for 5min for surface sterilization, then the seeds are soaked in 3% hydrogen peroxide for 2h, and finally the seeds are washed clean by sterile water. The sterilized seeds (8 seeds in each pot, the cotyledons grow out and then the seedlings are fixed, 3 seedlings are remained in each pot) are planted in a flowerpot (nutrient soil: vermiculite is 2:1), and the flowerpot is placed in a greenhouse with the temperature of 24 ℃, the light for 16h, the dark for 8h and the relative humidity of 70 percent for growth.

When cotton seedling cotyledons of upland cotton 86-1 of cotton susceptible variety are developed, EHA105 strain containing VIGS silencing carrier is taken, cultured at 28 ℃ to logarithmic phase, centrifuged at 6000 Xg for 5min, thallus is collected, and acetosyringone solution (10 mmoL/L2-morpholinoethanesulfonic acid (2- (N-morpholino) ethanesulfonic acid, MES),200 mu moL/L acetosyringone (As), 10mmoL/L MgCl₂) Resuspending the cells and adjusting the concentration to OD₆₀₀About 1.0. They were mixed with pCLCrVB-containing strain 1:1, respectively, and left to stand at room temperature for 3 hours before being used for cotton inoculation. Simultaneously, EHA105 strains containing pCLCrVA and pCLCrVB were mixed as empty vector controls. 1 piece of a 1mL disposable syringe is taken, the bacterial liquid is sucked, and the inoculation is carried out by injecting on the back of the cotyledon. Placing the inoculated cotton plant in an incubator, and culturing at the day and night temperature of 25 ℃ and 20 ℃ for 16h under illumination and 8h in darkness. And 17d, collecting leaves, extracting total RNA, detecting the gene expression quantity by using a fluorescent quantitative PCR technology, and biologically repeating for 3 times.

Inoculating Verticillium dahliae V991 (spore concentration 10) by root dipping method⁷spore/mL), 6 days after inoculation, the Disease of true leaves of the plants was investigated, and Disease Index (DI) was calculated. Disease index [ ∑ (number of disease plants at each stage × corresponding disease stage)/number of total investigated plants × highest disease stage (4)]X 100. The resistance rating is divided into 5 grades: the degree of infection of the plants from light to severe is represented by a scale of 0 to 4, and the plants are asymptomatic, yellow-green leaves, yellow leaves, mild leaf wilting and complete leaf wilting (complete death), respectively.

4.2 results of the experiment

The qRT-PCR technology is used for detecting the expression level of the target gene, and the result shows (as shown in figure 4), compared with a cotton plant inoculated with an empty vector pCLCrV (A + B), the expression level of the target gene of a plant silencing GhWRKY74 is obviously reduced (p is less than 0.05), the silencing efficiency is about 53%, and the VIGS technology is used for successfully silencing the gene.

The roots of both wild type and transgenic plants growing in the vermiculite culture flowerpot containing nutrient soil can grow well. After the cotton seedling grows for 20 days, suspension of V991 spore of strong pathogenic pathogen of deciduous leaf type (concentration is 10)⁷seed/mL), inoculating the germs by a root dipping method, and investigating the pathogenesis of the verticillium wilt after 5 days, 10 days and 15 days of inoculation.

The results show that: silencing the GhWRKY74 gene can significantly improve the disease resistance of plants, thereby enhancing the verticillium wilt resistance (as shown in figure 1).

The morbidity and disease index are shown in Table 17, the GhWRKY74 of the cotton plant 86-1 in the susceptible variety is silenced, the verticillium wilt disease index is only 54.6 +/-1.5, and is extremely obviously lower than the unloaded disease index 94.1 +/-2.1 and the wild type disease index 92.3 +/-3.2. The capital letters in table 1 represent significant differences at the 1% level.

TABLE 17 comparison of 86-1 verticillium wilt index in cotton plants of upland cotton varieties with different treatments

Therefore, after the gene GhWRKY74 is silenced by adopting VIGS, the disease resistance of the verticillium wilt-infected variety is improved under the stress of verticillium wilt; the wild type verticillium wilt-susceptible variety and the empty vector-transferred verticillium wilt-susceptible cotton variety are still highly susceptible to verticillium wilt bacteria under the stress of the verticillium wilt bacteria. The GhWRKY74 gene is proved to have the ability of reducing verticillium wilt, and the silencing of the GhWRKY74 gene can improve the verticillium wilt resistance of infected cotton varieties.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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aacaccgtcc atactatgag taggggaagt ggggttagtg tgcatcaaca tacctaatat 1500

ggggtttcaa actaaattta agctggtaga ttaacattgt tcttatatat atatatataa 1560

agagagagag agagaatagg aattgtgaac ttttttgaac tttgaatttg tgtaacccta 1620

tgtttcatct taatgttttt ggatttgtaa ggttgattgt atgtatgatt tgtggtgaca 1680

ttgtagaagg aaaactacaa aggttatcca aaatcatgta acatgaaatt cagcagtgcc 1740

aaaagaacta tgctgctatg aattgatttc ctaattttct taagtt 1786

<210> 2

<211> 326

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Phe Leu Ser Met Glu Lys Val Glu Glu Ala Asn Lys Ala Ala Ile

1 5 10 15

Glu Ser Cys Asn Arg Val Leu Ser Leu Leu Ser Leu Pro Lys Asp Gln

20 25 30

Leu Gln Tyr Ser Asn Leu Met Met Lys Thr Ser Glu Ala Val Phe Lys

35 40 45

Phe Lys Lys Val Val Ser Leu Leu Asn Asn Asp Leu Ser His Ser Arg

50 55 60

Val Arg Lys Ser Lys Met Phe Arg Ser Ser Ser Pro Gln Asn Ile Phe

65 70 75 80

Leu Glu Ser Pro Asn Cys Arg Thr Ile Leu Ser Pro Lys Pro Leu Gln

85 90 95

Val Tyr Pro Ser Asn Leu Phe Gln Lys Pro Pro Leu Glu Val Lys Pro

100 105 110

Ser Gln Asp Phe Ser Phe Val His Leu Gln Gln Gln Gln Gln Gln Met

115 120 125

Gln Gln Arg Leu Gln Phe Gln Gln Gln Gln Gln Gln Met Lys Tyr His

130 135 140

Ala Asp Met Val Phe Gly Lys Ser Asn Ser Gly Ile Asn Leu Lys Phe

145 150 155 160

Asp Gly Ser Ser Cys Thr Pro Thr Met Ser Ser Ala Gly Ser Phe Val

165 170 175

Ser Ser Leu Ser Leu Asp Gly Ser Val Ala Asn Leu Asp Gly Asn Ser

180 185 190

Phe His Leu Ile Gly Met Pro His His Phe Gly His Ile Ser Gln His

195 200 205

Ser Arg Lys Arg Cys Ser Gly Lys Gly Gln Asp Gly Ser Met Lys Cys

210 215 220

Gly Thr Ser Gly Lys Cys His Cys Ser Lys Arg Arg Lys Leu Arg Ile

225 230 235 240

Lys Arg Ser Ile Lys Val Pro Ala Ile Ser Asn Lys Val Ala Asp Ile

245 250 255

Pro Pro Asp Glu Tyr Ser Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys

260 265 270

Gly Ser Pro His Pro Arg Gly Tyr Tyr Lys Cys Ser Ser Val Arg Gly

275 280 285

Cys Pro Ala Arg Lys His Val Glu Arg Cys Leu Glu Asp Pro Ser Met

290 295 300

Leu Ile Val Thr Tyr Glu Gly Glu His Asn His Ser Arg Leu Leu Ser

305 310 315 320

Thr His Pro Ala His Thr

325

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtttttaa gtatggagaa ggttg 25

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcatgtatgc gcagggtg 18

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taccgtcgtt ccactagtga ttt 23

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtttggga atatgcagat cgga 24

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgcggatcct ccactagtga tttcactata gg 32

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgataatgcc cttatggagg atag 24

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gattacgcca agctttcaag gggtggtttt tggaacagg 39

<210> 10

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gattacgcca agctttgttt gcttcttcaa ccttctccat ac 42

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gaagtttttg tttgtttgtt tgtttttatt 30

<210> 13

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aacttaagaa aattaggaaa tcaattcata gc 32

Claims

1. The application of a greensickness-induced upland cotton related gene GhWRKY74 in cultivation of crop verticillium wilt-resistant varieties is characterized in that the gene expression is reduced by VIGS silencing the GhWRKY74 gene, so that the verticillium wilt resistance of greensickness-induced upland cotton is improved; the nucleotide sequence of the GhWRKY74 is shown as SEQ ID NO:1 is shown.

2. The use of the protein encoded by the upland cotton verticillium wilt-related gene GhWRKY74 in claim 1 in verticillium wilt resistance is characterized in that VIGS (virs) silences the GhWRKY74 gene, reduces the protein level expressed by the gene and improves the verticillium wilt resistance of upland cotton infected with verticillium wilt; the amino acid sequence of the protein coded by the GhWRKY74 is shown as SEQ ID NO:2, respectively.